RESUMO
Olfactory ensheathing cells (OECs) are specialized glial cells of the olfactory system, believed to play a role in the continuous production of olfactory neurons and ensheathment of their axons. Although OECs are used in therapeutic applications, little is known about the cellular mechanisms underlying their migratory behavior. Recently, we showed that OEC migration is sensitive to ganglioside blockage through A2B5 and Jones antibody in OEC culture. Gangliosides are common components of lipid rafts, where they participate in several cellular mechanisms, including cell migration. Here, we characterized OEC lipid rafts, analyzing the presence of specific proteins and gangliosides that are commonly expressed in motile neural cells, such as young neurons, oligodendrocyte progenitors, and glioma cells. Our results showed that lipid rafts isolated from OECs were enriched in cholesterol, sphingolipids, phosphatidylcholine, caveolin-1, flotillin-1, gangliosides GM1 and 9-O-acetyl GD3, A2B5-recognized gangliosides, CNPase, α-actinin, and ß1-integrin. Analysis of the actin cytoskeleton of OECs revealed stress fibers, membrane spikes, ruffled membranes and lamellipodia during cell migration, as well as the distribution of α-actinin in membrane projections. This is the first description of α-actinin and flotillin-1 in lipid rafts isolated from OECs and suggests that, together with ß1-integrin and gangliosides, membrane lipid rafts play a role during OEC migration. This study provides new information on the molecular composition of OEC membrane microdomains that can impact on our understanding of the role of OEC lipid rafts under physiological and pathological conditions of the nervous system, including inflammation, hypoxia, aging, neurodegenerative diseases, head trauma, brain tumor, and infection.
Assuntos
Microdomínios da Membrana/metabolismo , Bulbo Olfatório/citologia , 2',3'-Nucleotídeo Cíclico Fosfodiesterases/metabolismo , Animais , Biomarcadores/metabolismo , Células Cultivadas , Colesterol/metabolismo , Proteínas do Citoesqueleto/metabolismo , Gangliosídeos/metabolismo , Microdomínios da Membrana/ultraestrutura , Ratos Wistar , Proteínas S100/metabolismoRESUMO
The effect of cholesterol and ergosterol on supported lipid bilayers composed of 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and egg sphingomyelin (eSM) in a 1/1â¯M ratio was studied using atomic force microscopy. The addition of ergosterol or cholesterol to these membranes considerably modifies both the structure and the dynamics of the domains present in them. The height of the eSM enriched domains increases with concentration of both sterols, but more markedly with ergosterol. The height of the POPC enriched domains increases with concentration in a similar manner for both sterols. This effect is larger for eSM than for POPC when ergosterol, not cholesterol, is present. Domain coverage increases with both sterols at 5â¯mol% but decreases at 20â¯mol% and almost disappears at 40â¯mol%. The size of the eSM enriched domains decreases with sterol concentration, more markedly with cholesterol. Bilayer rupture forces show that overall stiffness increases with the addition of 5â¯mol% cholesterol, but only for the eSM enriched domains with ergosterol at the same concentration. At larger sterol concentrations the stiffness of both regions becomes reduced. At 40â¯mol% sterol concentration, both membranes present the same rupture force value. To gain mechanistic insight into these observations we performed Quantum Mechanical calculations and Molecular Dynamics simulations of the sterol molecules. We found that conformational freedom for the sterol molecules is quite different. This difference might be behind the observed phenomena. Finally, the different action of sterols on membrane properties is related to the sterol-dependent ionophoretic activity of polyene antibiotics.
Assuntos
Colesterol/química , Ergosterol/química , Bicamadas Lipídicas/química , Microdomínios da Membrana/química , Microdomínios da Membrana/ultraestrutura , Fosfatidilcolinas/química , Esfingomielinas/química , Lipossomas Unilamelares/químicaRESUMO
The phase diagram of mixed monolayers composed of dimyristoyl phosphatidylcholine (DMPC) and stearic acid (SA) on different subphases was previously reported. It was observed that on acid subphases, liquid-condensed domains with shapes that depend on the SA proportion are formed. For mixtures with 40-45mole% of SA, the domain shape changes from flower-like to circular domains. In this work, we carried out a detailed study of the driving force for the shape change. We find that it is related to the domain density which, in turn, is driven by the domain nucleation process and thus by oversaturation of the system leading to phase segregation. This could be a way of self-regulating the local electrostatics and mechanical properties in membrane surfaces with segregated phase domains.
Assuntos
Dimiristoilfosfatidilcolina/química , Microdomínios da Membrana/ultraestrutura , Ácidos Esteáricos/química , Microdomínios da Membrana/química , Transição de FaseRESUMO
Microdomains, or lipid rafts, are transient membrane regions enriched in sphingolipids and sterols that have only recently, but intensively, been studied in plants. In this work, we report a detailed, easy-to-follow, and fast procedure to isolate detergent-resistant membranes (DRMs) from purified plasma membranes (PMs) that was used to obtain DRMs from Phaseolus vulgaris and Nicotiana tabacum leaves and germinating Zea mays embryos. Characterized according to yield, ultrastructure, and sterol composition, these DRM preparations showed similarities to analogous preparations from other eukaryotic cells. Isolation of DRMs from germinating maize embryos reveals the presence of microdomains at very early developmental stages of plants.
Assuntos
Microdomínios da Membrana/química , Nicotiana/química , Phaseolus/química , Fotossíntese , Zea mays/química , Detergentes/química , Microdomínios da Membrana/ultraestrutura , Phaseolus/ultraestrutura , Sementes/química , Sementes/ultraestrutura , Esteróis/análise , Esteróis/química , Nicotiana/ultraestrutura , Zea mays/ultraestruturaRESUMO
Intrauterine growth restriction (IUGR) and preeclampsia (PE) are leading causes of perinatal and maternal morbidity and mortality. Previously we reported the expression of lipid rafts in classical microvillous membrane (MVM) and light microvillous membrane (LMVM), two subdomains in apical membrane from the human placental syncytiotrophoblast (hSTB), which constitute the epithelium responsible for maternal-fetal transport. Here the aim was to study the raft and cytoskeletal proteins from PE and IUGR. Microdomains from MVM and LMVM were tested with raft markers (placental alkaline phosphatase, lipid ganglioside, and annexin 2) and a nonraft marker (hTf-R). No changes were detected with those markers in whole purified apical membranes in normal, PE, and IUGR pregnancies; however, their patterns of distribution in lipid rafts were different in PE and IUGR. Cholesterol depletion modified their segregation, confirming their presence in lipid rafts, although unlike normal placenta, in these pathologies there is only one type of microdomain. Additionally, the cytoskeleton proteins actin, ezrin, and cytokeratin-7 showed clear differences between normal and pathological membranes. Cytokeratin-7 expression decreased to 50% in PE, and the distribution between LMVM and MVM (~43 and 57%, respectively) changed in both PE and IUGR, in contrast with the asymmetrical enrichment obtained in normal LMVM (~62%). In conclusion, lipid rafts from IUGR and PE have different features compared to rafts from normal placentae, and this is associated with alterations in the expression and distribution of cytoskeletal proteins.
Assuntos
Retardo do Crescimento Fetal/metabolismo , Microdomínios da Membrana , Microvilosidades/metabolismo , Placenta/metabolismo , Pré-Eclâmpsia/metabolismo , Trofoblastos/metabolismo , Actinas/genética , Actinas/metabolismo , Biomarcadores/análise , Western Blotting , Estudos de Casos e Controles , Fracionamento Celular , Colesterol/metabolismo , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Eletroforese em Gel de Poliacrilamida , Feminino , Retardo do Crescimento Fetal/patologia , Expressão Gênica , Humanos , Queratina-7/genética , Queratina-7/metabolismo , Microdomínios da Membrana/química , Microdomínios da Membrana/genética , Microdomínios da Membrana/metabolismo , Microdomínios da Membrana/ultraestrutura , Microscopia Confocal , Microvilosidades/patologia , Especificidade de Órgãos , Placenta/patologia , Pré-Eclâmpsia/patologia , Gravidez , Trofoblastos/patologiaRESUMO
The structural organization of the plasma membrane of eukaryotic cells is briefly revised taking into consideration the organization of proteins and lipids and the concept of microdomains, lipid rafts and detergent resistant membranes. The biochemical data available concerning the presence of microdomains in parasitic protozoa is reviewed and emphasis is given on the identification of special domains recognized by morphological approaches, especially with the use of the freeze-fracture technique.
Assuntos
Membrana Celular/química , Eucariotos/ultraestrutura , Microdomínios da Membrana/química , Animais , Membrana Celular/efeitos dos fármacos , Membrana Celular/ultraestrutura , Detergentes/farmacologia , Eucariotos/química , Técnica de Fratura por Congelamento , Humanos , Microdomínios da Membrana/ultraestrutura , Microscopia EletrônicaRESUMO
Recent studies have shown that, in mast cells, membrane microdomains rich in cholesterol and glycosphingolipids called lipid rafts play an important role in FcepsilonRI signaling. The present study demonstrates that, in RBL-2H3 cells following stimulation, the mast cell-specific gangliosides associated with FcepsilonRI are internalized from lipid rafts along with the receptor. When the cells are labeled with iodinated antibodies against the gangliosides or against FcepsilonRI and the cell components are then fractionated on Percoll density gradients, in stimulated cells the gangliosides are internalized with the same kinetics as FcepsilonRI and at 3 hr are present in the dense lysosome fraction. Using transmission electron microscopy, with antibody against the gangliosides conjugated to horseradish peroxidase and antibody against FcepsilonRI conjugated to colloidal gold, it was possible to demonstrate that the gangliosides and FcepsilonRI are internalized in the same coated vesicles. At 5 min, the gangliosides and FcepsilonRI can be identified in early endosomes and at 3 hr are found together in acid phosphatase-positive lysosomes. This study demonstrates that the mast cell-specific gangliosides are internalized from lipid rafts in the same vesicles and traffic intracellularly with the same kinetics as FcepsilonRI. This study contains online supplemental material at http://www.jhc.org. Please visit this article online to view these materials.
Assuntos
Endocitose/fisiologia , Gangliosídeos/metabolismo , Microdomínios da Membrana/metabolismo , Receptores de IgE/metabolismo , Transdução de Sinais , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/imunologia , Linhagem Celular Tumoral , Invaginações Revestidas da Membrana Celular/metabolismo , Invaginações Revestidas da Membrana Celular/ultraestrutura , Gangliosídeos/imunologia , Coloide de Ouro/química , Coloide de Ouro/imunologia , Peroxidase do Rábano Silvestre/química , Peroxidase do Rábano Silvestre/imunologia , Cinética , Lisossomos/metabolismo , Lisossomos/ultraestrutura , Mastócitos/química , Microdomínios da Membrana/imunologia , Microdomínios da Membrana/ultraestrutura , Microscopia Eletrônica de Varredura , Microscopia ImunoeletrônicaRESUMO
The importance of apoptosis as a form of programmed cell death was recognized in the 1980s, whereas the central role of mitochondria in controlling this process was identifi ed in the mid-1990s. An important event in apoptosis is the collapse of the mitochondrial transmembrane potential (ÃØm), with the ensuing loss of the selective permeability of the inner membrane resulting in swelling of the hyperosmolar mitochondrial matrix. This event is known as the mitochondrial permeability transition (MPT). After swelling of the intermembrane space, the outer membrane ruptures, exposing the permeable inner membrane. An increasingly swollen matrix covered by the inner membrane eventually herniates into the cytoplasm through the breach formed in the outer membrane (OM). The increase in surface area of the inner mitochondrial membrane (IMM) involves the unfolding of membrane stored in the cristae. This membrane movement is osmotically driven since the cytoplasm has a lower osmolality. The proteins partly embedded in the inner membrane are thus exposed to the cytoplasm. In nine out of ten electron microscopy studies of isolated mitochondria expressing the permeability transition, the existing ruptures of the OMM were overlooked. The MPT can also be recognized in individual mitochondria by using fl uorescent probes that are not retained in these organelles once the ÃØm is lost. In cases in which there is no rupture of the OMM, cytochrome c must be released from mitochondria with impermeable inner membranes. Examination of several hundred of the more than 61,000 published papers on programmed cell death revealed that the key signaling events of apoptosis, such as the onset of the MPT, mitochondrial swelling and cytochrome c release to the cytoplasm, are infl uenced by factors such as the cell type and presence of apoptogenic agents...